3. Drinking Water Sectional Committee, FAD 25
FOREWORD
This Indian Standard (Second Revision) was adopted by the Bureau of Indian Standards, after the draft finalized
by the Drinking Water Sectional Committee had been approved by the Food and Agriculture Division Council.
This standard was originally published in 1983.A report prepared by the World Health Organization in cooperation
with the World Bank showed that in 1975, some 1 230 million people were without safe water supplies. These
appalling facts were central to the United Nations decision to declare an International Drinking Water Supply and
Sanitation decade, beginning in 1981. Further, the VI Five-Year Plan of India had made a special provision for
availability of safe drinking water for the masses. Therefore, the standard was formulated with the objective of
assessing the quality of water resources, and to check the effectiveness of water treatment and supply by the
concerned authorities.
The first revision was undertaken to take into account the up-to-date information available about the nature and
effect of various contaminants as also the new techniques for identifying and determining their concentration.
Based on experience gained additional requirements for alkalinity; aluminium and boron were incorporated and
the permissible limits for dissolved solids, nitrate and pesticides residues modified.
As per the eleventh five year plan document of India (2007-12), there are about 2.17 lakh quality affected habitations
in the country with more than half affected with excess iron, followed by fluoride, salinity, nitrate and arsenic in
that order. Further, approximately, 10 million cases of diarrhoea, more than 7.2 lakh typhoid cases and 1.5 lakh
viral hepatitis cases occur every year a majority of which are contributed by unclean water supply and poor
sanitation. The eleventh five year plan document of India (2007-2012) recognizes dealing with the issue of water
quality as a major challenge and aims at addressing water quality problems in all quality affected habitations with
emphasis on community participation and awareness campaigns as well as on top most priority to water quality
surveillance and monitoring by setting up of water quality testing laboratories strengthened with qualified
manpower, equipments and chemicals.
The second revision was undertaken to upgrade the requirements of the standard and align with the internationally
available specifications on drinking water. In this revision assistance has been derived from the following:
a) EU Directives relating to the quality of water intended for human consumption (80/778/EEC) and Council
Directive 98/83/EC.
b) USEPA standard — National Primary Drinking Water Standard. EPA 816-F-02-013 dated July, 2002.
c) WHO Guidelines for Drinking Water Quality. 3rd Edition Vol. 1 Recommendations, 2008.
d) Manual on Water Supply and Treatment, third edition — revised and updated May 1999, Ministry of
Urban Development, New Delhi.
This standard specifies the acceptable limits and the permissible limits in the absence of alternate source. It is
recommended that the acceptable limit is to be implemented as values in excess of those mentioned under
‘Acceptable’render the water not suitable. Such a value may, however, be tolerated in the absence of an alternative
source. However, if the value exceeds the limits indicated under ‘permissible limit in the absence of alternate
source’ in col 4 of Tables 1 to 4, the sources will have to be rejected.
Pesticide residues limits and test methods given in Table 5 are based on consumption pattern, persistence and
available manufacturing data. The limits have been specified based on WHO guidelines, wherever available. In
cases where WHO guidelines are not available, the standards available from other countries have been examined
and incorporated, taking in view the Indian conditions.
In this revision, additional requirements for ammonia, chloramines, barium, molybdenum, silver, sulphide, nickel,
polychlorinated biphenyls and trihalomethanes have been incorporated while the requirements for colour, turbidity,
total hardness, free residual chlorine, iron, magnesium, mineral oil, boron, cadmium, total arsenic, lead, polynuclear
aromatic hydrocarbons, pesticides and bacteriological requirements have been modified.
In this revision, requirement and test method for virological examination have been included. Further, requirements
and test methods for cryptosporidium and giardia have also been specified.
Routine surveillance of drinking water supplies should be carried out by the relevant authorities to understand
the risk of specific pathogens and to define proper control procedures. The WHO Guidelines for Drinking Water
Quality, 3rd Edition, Vol. 1 may be referred for specific recommendations on using a water safety approach
incorporating risk identification. Precautions/Care should be taken to prevent contamination of drinking water
from chlorine resistant parasites such as cryptosporidium species and giardia.
4. 1
IS 10500 : 2012
Indian Standard
DRINKING WATER — SPECIFICATION
( Second Revision )
1 SCOPE
This standard prescribes the requirements and the
methods of sampling and test for drinking water.
2 REFERENCES
The standards listed in Annex A contain provisions
which through reference in this text, constitute
provisions of this standard. At the time of publication,
the editions indicated were valid. All standards are
subject to revision and parties to agreements based on
this standard are encouraged to investigate the
possibility of applying the most recent editions of the
standards indicated in Annex A.
3 TERMINOLOGY
For the purpose of this standard the following definition
shall apply.
3.1 Drinking Water — Drinking water is water
intended for human consumption for drinking and
cooking purposes from any source. It includes water
(treated or untreated) supplied by any means for human
consumption.
4 REQUIREMENTS
Drinking water shall comply with the requirements
given in Tables 1 to 4. The analysis of pesticide residues
given in Table 3 shall be conducted by a recognized
laboratory using internationally established test method
meeting the residue limits as given in Table 5.
Drinking water shall also comply with bacteriological
requirements (see 4.1), virological requirements
(see 4.2) and biological requirements (see 4.3).
4.1 Bacteriological Requirements
4.1.1 Water in Distribution System
Ideally, all samples taken from the distribution system
including consumers’ premises, should be free from
coliform organisms and the following bacteriological
quality of drinking water collected in the distribution
system, as given in Table 6 is, therefore specified when
tested in accordance with IS 1622.
4.2 Virological Requirements
4.2.1 Ideally, all samples taken from the distribution
Table 1 Organoleptic and Physical Parameters
(Foreword and Clause 4)
Sl No. Characteristic Requirement Permissible Method of Test, Remarks
(Acceptable Limit in the Ref to Part of
Limit) Absence of IS 3025
Alternate
Source
(1) (2) (3) (4) (5) (6)
i) Colour, Hazen units, Max 5 15 Part 4 Extended to 15 only, if toxic substances
are not suspected in absence of alter-
nate sources
ii) Odour Agreeable Agreeable Part 5 a) Test cold and when heated
b) Test at several dilutions
iii) pH value 6.5-8.5 No relaxation Part 11 —
iv) Taste Agreeable Agreeable Parts 7 and 8 Test to be conducted only after safety
has been established
v) Turbidity, NTU, Max 1 5 Part 10 —
vi) Total dissolved solids, mg/l, 500 2 000 Part 16 —
Max
NOTE — It is recommended that the acceptable limit is to be implemented. Values in excess of those mentioned under ‘acceptable’
render the water not suitable, but still may be tolerated in the absence of an alternative source but up to the limits indicated under
‘permissible limit in the absence of alternate source’ in col 4, above which the sources will have to be rejected.
5. 2
IS 10500 : 2012
Table 2 General Parameters Concerning Substances Undesirable in Excessive Amounts
(Foreword and Clause 4)
Sl No. Characteristic Requirement Permissible Method of Test, Remarks
(Acceptable Limit in the Ref to
Limit) Absence of
Alternate
Source
(1) (2) (3) (4) (5) (6)
i) Aluminium (as Al), mg/l, Max 0.03 0.2 IS 3025 (Part 55) —
ii) Ammonia (as total ammonia-N), 0.5 No relaxation IS 3025 (Part 34) —
mg/l, Max
iii) Anionic detergents (as MBAS) 0.2 1.0 Annex K of IS 13428 —
mg/l, Max
iv) Barium (as Ba), mg/l, Max 0.7 No relaxation Annex F of IS 13428* —
or IS 15302
v) Boron (as B), mg/l, Max 0.5 1.0 IS 3025 (Part 57) —
vi) Calcium (as Ca), mg/l, Max 75 200 IS 3025 (Part 40) —
vii) Chloramines (as Cl2
), mg/l, Max 4.0 No relaxation IS 3025 (Part 26)* —
or APHA 4500-Cl G
viii) Chloride (as Cl), mg/l, Max 250 1 000 IS 3025 (Part 32) —
ix) Copper (as Cu), mg/l, Max 0.05 1.5 IS 3025 (Part 42) —
x) Fluoride (as F) mg/l, Max 1.0 1.5 IS 3025 (Part 60) —
xi) Free residual chlorine, mg/l, Min 0.2 1 IS 3025 (Part 26) To be applicable only when
water is chlorinated. Tested
at consumer end. When pro-
tection against viral infec-
tion is required, it should be
minimum 0.5 mg/l
xii) Iron (as Fe), mg/l, Max 0.3 No relaxation IS 3025 (Part 53) Total concentration of man-
ganese (as Mn) and iron (as
Fe) shall not exceed 0.3 mg/l
xiii) Magnesium (as Mg), mg/l, Max 30 100 IS 3025 (Part 46) —
xiv) Manganese (as Mn), mg/l, Max 0.1 0.3 IS 3025 (Part 59) Total concentration of man-
ganese (as Mn) and iron (as
Fe) shall not exceed 0.3 mg/l
xv) Mineral oil, mg/l, Max 0.5 No relaxation Clause 6 of IS 3025 —
(Part 39) Infrared
partition method
xvi) Nitrate (as NO3
), mg/l, Max 45 No relaxation IS 3025 (Part 34) —
xvii) Phenolic compounds (as C6
H5
OH), 0.001 0.002 IS 3025 (Part 43) —
mg/l, Max
xviii) Selenium (as Se), mg/l, Max 0.01 No relaxation IS 3025 (Part 56) or —
IS 15303*
xix) Silver (as Ag), mg/l, Max 0.1 No relaxation Annex J of IS 13428 —
xx) Sulphate (as SO4
) mg/l, Max 200 400 IS 3025 (Part 24) May be extended to 400 pro-
vided that Magnesium does
not exceed 30
xxi) Sulphide (as H2
S), mg/l, Max 0.05 No relaxation IS 3025 (Part 29) —
xxii) Total alkalinity as calcium 200 600 IS 3025 (Part 23) —
carbonate, mg/l, Max
xxiii) Total hardness (as CaCO3
), 200 600 IS 3025 (Part 21) —
mg/l, Max
xxiv) Zinc (as Zn), mg/l, Max 5 15 IS 3025 (Part 49) —
NOTES
1 In case of dispute, the method indicated by '*' shall be the referee method.
2 It is recommended that the acceptable limit is to be implemented. Values in excess of those mentioned under ‘acceptable’ render the
water not suitable, but still may be tolerated in the absence of an alternative source but up to the limits indicated under ‘permissible
limit in the absence of alternate source’ in col 4, above which the sources will have to be rejected.
6. 3
IS 10500 : 2012
Table 3 Parameters Concerning Toxic Substances
(Foreword and Clause 4)
Sl No. Characteristic Requirement Permissible Method of Test, Remarks
(Acceptable Limit in the Ref to
Limit) Absence of
Alternate
Source
(1) (2) (3) (4) (5) (6)
i) Cadmium (as Cd), mg/l, Max 0.003 No relaxation IS 3025 (Part 41) —
ii) Cyanide (as CN), mg/l, Max 0.05 No relaxation IS 3025 (Part 27) —
iii) Lead (as Pb), mg/l, Max 0.01 No relaxation IS 3025 (Part 47) —
iv) Mercury (as Hg), mg/l, Max 0.001 No relaxation IS 3025 (Part 48)/ —
Mercury analyser
v) Molybdenum (as Mo), mg/l, Max 0.07 No relaxation IS 3025 (Part 2) —
vi) Nickel (as Ni), mg/l, Max 0.02 No relaxation IS 3025 (Part 54) —
vii) Pesticides, µg/l, Max See Table 5 No relaxation See Table 5 —
viii) Polychlorinated biphenyls, mg/l, 0.000 5 No relaxation ASTM 5175* —
Max or APHA 6630
ix) Polynuclear aromatic hydro- 0.000 1 No relaxation APHA 6440 —
carbons (as PAH), mg/l, Max
x) Total arsenic (as As), mg/l, Max 0.01 0.05 IS 3025 (Part 37) —
xi) Total chromium (as Cr), mg/l, Max 0.05 No relaxation IS 3025 (Part 52) —
xii) Trihalomethanes:
a) Bromoform, mg/l, Max 0.1 No relaxation ASTM D 3973-85* —
or APHA 6232
b) Dibromochloromethane, 0.1 No relaxation ASTM D 3973-85* —
mg/l, Max or APHA 6232
c) Bromodichloromethane, 0.06 No relaxation ASTM D 3973-85* —
mg/l, Max or APHA 6232
d) Chloroform, mg/l, Max 0.2 No relaxation ASTM D 3973-85* —
or APHA 6232
NOTES
1 In case of dispute, the method indicated by '*' shall be the referee method.
2 It is recommended that the acceptable limit is to be implemented. Values in excess of those mentioned under ‘acceptable’ render the
water not suitable, but still may be tolerated in the absence of an alternative source but up to the limits indicated under 'permissible
limit in the absence of alternate source’ in col 4, above which the sources will have to be rejected.
Table 4 Parameters Concerning Radioactive Substances
(Foreword and Clause 4)
Sl No. Characteristic Requirement Permissible Method of Test, Remarks
(Acceptable Limit in the Ref to Part of
Limit) Absence of IS 14194
Alternate
Source
(1) (2) (3) (4) (5) (6)
i) Radioactive materials:
a) Alpha emitters Bq/l, Max 0.1 No relaxation Part 2 —
b) Beta emitters Bq/l, Max 1.0 No relaxation Part 1 —
NOTE — It is recommended that the acceptable limit is to be implemented. Values in excess of those mentioned under ‘acceptable’
render the water not suitable, but still may be tolerated in the absence of an alternative source but up to the limits indicated under
‘permissible limit in the absence of alternate source’ in col 4, above which the sources will have to be rejected.
7. 4
IS 10500 : 2012
Table 5 Pesticide Residues Limits and Test Method
(Foreword and Table 3)
Sl No. Pesticide Limit Method of Test, Ref to
µg/l
USEPA AOAC/ ISO
(1) (2) (3) (4) (5)
i) Alachlor 20 525.2, 507 —
ii) Atrazine 2 525.2, 8141 A —
iii) Aldrin/ Dieldrin 0.03 508 —
iv) Alpha HCH 0.01 508 —
v) Beta HCH 0.04 508 —
vi) Butachlor 125 525.2, 8141 A —
vii) Chlorpyriphos 30 525.2, 8141 A —
viii) Delta HCH 0.04 508 —
ix) 2,4- Dichlorophenoxyacetic acid 30 515.1 —
x) DDT (o, p and p, p – Isomers of DDT, 1 508 AOAC 990.06
DDE and DDD)
xi) Endosulfan (alpha, beta, and sulphate) 0.4 508 AOAC 990.06
xii) Ethion 3 1657 A —
xiii) Gamma — HCH (Lindane) 2 508 AOAC 990.06
xiv) Isoproturon 9 532 —
xv) Malathion 190 8141 A —
xvi) Methyl parathion 0.3 8141 A ISO 10695
xvii) Monocrotophos 1 8141 A —
xviii) Phorate 2 8141 A —
NOTE — Test methods are for guidance and reference for testing laboratory. In case of two methods, USEPA method shall be the
reference method.
Table 6 Bacteriological Quality of Drinking Water1)
(Clause 4.1.1)
Sl No. Organisms Requirements
(1) (2) (3)
i) All water intended for drinking:
a) E. coli or thermotolerant coliform bacteria2), 3)
Shall not be detectable in any 100 ml sample
ii) Treated water entering the distribution system:
a) E. coli or thermotolerant coliform bacteria2)
Shall not be detectable in any 100 ml sample
b) Total coliform bacteria Shall not be detectable in any 100 ml sample
iii) Treated water in the distribution system:
a) E. coli or thermotolerant coliform bacteria Shall not be detectable in any 100 ml sample
b) Total coliform bacteria Shall not be detectable in any 100 ml sample
1)
Immediate investigative action shall be taken if either E.coli or total coliform bacteria are detected. The minimum action in the case of
total coliform bacteria is repeat sampling; if these bacteria are detected in the repeat sample, the cause shall be determined by immediate
further investigation.
2)
Although, E. coli is the more precise indicator of faecal pollution, the count of thermotolerant coliform bacteria is an acceptable alternative.
If necessary, proper confirmatory tests shall be carried out. Total coliform bacteria are not acceptable indicators of the sanitary quality of
rural water supplies, particularly in tropical areas where many bacteria of no sanitary significance occur in almost all untreated supplies.
3)
It is recognized that, in the great majority of rural water supplies in developing countries, faecal contamination is widespread. Under
these conditions, the national surveillance agency should set medium-term targets for progressive improvement of water supplies.
8. 5
IS 10500 : 2012
system including consumers’ premises, should be free
from virus.
4.2.2 None of the generally accepted sewage treatment
methods yield virus-free effluent. Although a number
of investigators have found activated sludge treatment
to be superior to trickling filters from this point of view,
it seems possible that chemical precipitation methods
will prove to be the most effective.
4.2.3 Virus can be isolated from raw water and from
springs, enterovirus, reovirus, and adenovirus have
been found in water, the first named being the most
resistant to chlorination. If enterovirus are absent from
chlorinated water, it can be assumed that the water is
safe to drink. Some uncertainty still remains about the
virus of infectious hepatitis, since it has not so far been
isolated but in view of the morphology and resistance
of enterovirus it is likely that, if they have been
inactivated hepatitis virus will have been inactivated
also.
4.2.4 An exponential relationship exists between the
rate of virus inactivation and the redox potential. A
redox potential of 650 mV (measured between
platinum and calomel electrodes) will cause almost
instantaneous inactivation of even high concentrations
of virus. Such a potential can be obtained with even a
low concentration of free chlorine, but only with an
extremely high concentration of combined chlorine.
This oxidative inactivation may be achieved with a
number of other oxidants also, for example, iodine,
ozone and potassium permanganate, but the effect of
the oxidants will always be counteracted, if reducing
components, which are mainly organic, are present.
As a consequence, the sensitivity of virus towards
disinfectants will depend on the milieu just as much as
on the particular disinfectant used.
4.2.5 Viruses are generally resistant to disinfectants as
well as get protected on account of presence of
particulate and organic matter in water. Because the
difference between the resistance of coliform
organisms and of virus to disinfection by oxidants
increases with increasing concentration of reducing
components, for example, organic matter, it cannot be
assumed that the absence of available coliform
organisms implies freedom from active virus under
circumstances where a free chlorine residual cannot
be maintained. Sedimentation and slow sand filtration
in themselves may contribute to the removal of virus
from water.
4.2.6 In practice, >0.5 mg/l of free chlorine for 1 h is
sufficient to inactivate virus, even in water that was
originally polluted provided the water is free from
particulates and organic matter.
4.2.7 MS2 phage are indicator of viral contamination
in drinking water. MS2 phage shall be absent in 1 litre
of water when tested in accordance with USEPA
method 1602. If MS2 phage are detected in the drinking
water, virological examination shall be done by the
Polymerase Chain Reaction (PCR) method for
virological examination as given in Annex B. USEPA
method in Manual of Method forVirology Chapter 16,
June 2001 shall be the alternate method. If viruses are
detected, the cause shall be determined by immediate
further investigation.
4.3 Biological Requirements
4.3.1 Ideally, all samples taken including consumers
premises should be free from biological organisms.
Biological examination is of value in determining the
causes of objectionable tastes and odours in water and
controlling remedial treatments, in helping to interpret
the results of various chemical analysis, and in
explaining the causes of clogging in distribution pipes
and filters. In some instances, it may be of use in
demonstrating that water from one source has been
mixed with that from another.
4.3.2 The biological qualities of water are of greater
importance when the supply has not undergone the
conventional flocculation and filtration processes, since
increased growth of methane-utilizing bacteria on
biological slimes in pipes may then be expected, and
the development of bryozoal growths such as
Plumatella may cause operational difficulties.
4.3.3 Some of the animalcules found in water mains
may be free-living in the water, but others such as
Dreissena and Asellus are more or less firmly attached
to the inside of the mains. Although these animalcules
are not themselves pathogenic, they may harbour
pathogenic organisms or virus in their intestines, thus
protecting these pathogens from destruction by
chlorine.
4.3.4 Chlorination, at the dosages normally employed
in waterworks, is ineffective against certain parasites,
including amoebic cysts; they can be excluded only
by effective filtration or by higher chlorine doses than
can be tolerated without subsequent dechlorination.
Amoebiasis can be conveyed by water completely free
from enteric bacteria; microscopic examination after
concentration is, therefore, the only safe method of
identification.
4.3.5 Strict precautions against back-syphonage and
cross-connections are required, if amoebic cysts are
found in a distribution system containing tested water.
4.3.6 The cercariae of schistosomiasis can be detected
by similar microscopic examination, but there is, in
9. 6
IS 10500 : 2012
any case, no evidence to suggest that this disease is
normally spread through piped water supplies.
4.3.7 The cyclops vector of the embryos of
Dracunculus medinensis which causes dracontiasis or
Guinea-worm disease can be found in open wells in a
number of tropical areas. They are identifiable by
microscopic examination. Such well supplies are
frequently used untreated, but the parasite can be
relatively easily excluded by simple physical
improvements in the form of curbs, drainage, and apron
surrounds and other measures which prevent physical
contact with the water source.
4.3.8 Cryptosporidium shall be absent in 10 liter of
water when tested in accordance with USEPA method
1622 or USEPA method 1623* or ISO 15553 : 2006.
4.3.9 Giardia shall be absent in 10 liter of water when
tested in accordance with USEPA method 1623* or
ISO 15553 : 2006.
4.3.10 The drinking water shall be free from
microscopic organisms such as algae, zooplanktons,
flagellates, parasites and toxin producing organisms.
An illustrative (and not exhaustive) list is given in
Annex C for guidance.
NOTE — In case of dispute, the method indicated by ‘*’ in
4.3.8 and 4.3.9 shall be referee method.
5 SAMPLING
Representative samples of water shall be drawn as
prescribed in IS 1622 and IS 3025 (Part 1).
ANNEX A
(Clause 2)
LIST OF REFERRED INDIAN STANDARDS
IS No. Title
1622 : 1981 Methods of sampling and
microbiological examination of
water (first revision)
3025 Methods of sampling and test
(physical and chemical) for water and
waste water:
(Part 1) : 1987 Sampling (first revision)
(Part 2) : 2002 Determination of 33 elements by
inductively coupled plasma atomic
emission spectroscopy
(Part 4) : 1983 Colour (first revision)
(Part 5) : 1983 Odour (first revision)
(Part 7) : 1984 Taste threshold (first revision)
(Part 8) : 1984 Tasting rate (first revision)
(Part 10) : 1984 Turbidity (first revision)
(Part 11) : 1983 pH value (first revision)
(Part 16) : 1984 Filterable residue (total dissolved
solids) (first revision)
(Part 21) : 1983 Total hardness (first revision)
(Part 23) : 1983 Alkalinity (first revision)
(Part 24) : 1986 Sulphates (first revision)
(Part 26) : 1986 Chlorine residual (first revision)
(Part 27) : 1986 Cyanide (first revision)
(Part 29) : 1986 Sulphide (first revision)
(Part 32) : 1988 Chloride (first revision)
(Part 34) : 1988 Nitrogen (first revision)
(Part 37) : 1988 Arsenic (first revision)
(Part 39) : 1989 Oil and grease
(Part 40) : 1991 Calcium
IS No. Title
(Part 41) : 1992 Cadmium (first revision)
(Part 42) : 1992 Copper (first revision)
(Part 43) : 1992 Phenols (first revision)
(Part 46) : 1994 Magnesium
(Part 47) : 1994 Lead
(Part 48) : 1994 Mercury
(Part 49) : 1994 Zinc
(Part 52) : 2003 Chromium
(Part 53) : 2003 Iron
(Part 54) : 2003 Nickel
(Part 55) : 2003 Aluminium
(Part 56) : 2003 Selenium
(Part 57) : 2005 Boron
(Part 59) : 2006 Manganese
(Part 60) : 2008 Fluoride
13428 : 2003 Packaged natural mineral water —
Specification (first revision)
14194 Radionuclides in environmental
samples — Method of estimation:
(Part 1) : 1994 Gross beta activity measurement
(Part 2) : 1994 Gross alpha activity measurement
15302 : 2002 Determination of aluminium and
barium in water by direct nitrous
oxide-acetylene flame atomic
absorption spectrometry
15303 : 2002 Determination of antimony, iron and
selenium in water by electrothermal
atomic absorption spectrometry
10. 7
IS 10500 : 2012
B-1 GENERAL
The method involves the concentration of viruses from
100 litre of drinking water to 1 ml by membrane filter
technique.The concentrate is subjected to amplification
using polymerase chain reaction (PCR) and primers
based on highly conserved regions of viral genomes.
This method can detect as low as 10 genome copies.
Stringent precautions are needed to avoid
contamination with amplified DNA products leading
to false positive reactions. Detection of hepatitisA virus
(HAV) RNA and enterovirus (EV) RNA is considered
as an indication of presence of viruses in water. Steps
involved include concentration of water, RNA
extraction, complementary DNA (cDNA) synthesis and
PCR.
B-2 CONCENTRATION OF DRINKING WATER
B-2.1 Apparatus
B-2.1.1 Pressure Pump
B-2.1.2 Membrane Filter Assembly with 144 mm
Diameter with Tripod Stand
B-2.1.3 Pressure Vessel (50 litre capacity) with
Pressure Gauge
B-2.1.4 Inter-connecting Pressure Tubes
B-2.2 Reagents
Autoclaved double distilled water shall be used for the
preparation of reagents/buffers in this study.
B-2.2.1 Aluminium Chloride
B-2.2.2 HCl/NaOH Urea (Extra Pure)
B-2.2.3 Disodium Hydrogen Phosphate (Na2HPO4.
2H2O) — 0.2 M, filter sterilized.
B-2.2.4 Sodium Dihydrogen Phosphate (NaH2PO4.
2H2O) — 0.2 M, filter sterilized.
B-2.2.5 Citric Acid — 0.1 M, filter sterilized.
B-2.2.6 L-Arginine — 0.5 M, filter sterilized.
B-2.2.7 Urea-Arginine Phosphate Buffer (U-APB) —
Mix 4.5 g of urea with 2 ml of 0.2 M NaH2PO4 and
2 ml of 0.5 M L - Arginine and make up the volume to
50 ml with sterile distilled water. The pH of the eluent
shall be 9.0.
B-2.2.8 Magnesium Chloride (MgCl2) — 1 M.
B-2.2.9 McII Vaines Buffer (pH 5.0) — Mix 9.7 ml of
ANNEX B
(Clause 4.2.7)
POLYMERASE CHAIN REACTION (PCR) METHOD
0.1 M citric acid with 10.3 ml of 0.2 M Na2HPO4 .2H2O
under sterile conditions.
B-2.3 Procedure
Filter 100 litre of drinking water sample through
membrane filter assembly using either positively
charged membrane of 144 mm diameter or 0.22 micron
diameter pore size nitrocellulose membrane. For
positively charged membrane the test water pH need
not be adjusted. But for the 0.22 micron nitrocellulose
membrane adjust the pH to 3.5 after adding the
aluminium chloride as a coagulant to a final
concentration of 0.000 5 M.
At lower pH pass the water through the membrane.
The flow rate shall be 40 litre/h approximately. After
the completion of the filtration, elute the adsorbed
particles using 100 ml of urea-arginine phosphate
buffer (U-APB). Precipitate the suspended particles
using 1 ml of magnesium chloride (1 M). Dissolve the
resultant precipitate centrifuged out of the sample in
800-1.0 ml of McII vaines buffer. The processed sample
can be stored at refrigerator until required.
B-3 RNA EXTRACTION
B-3.1 Apparatus
B-3.1.1 Cooling Centrifuge
B-3.1.2 Deep Freezer (–20°C)
B-3.1.3 Vortex Mixer
B-3.1.4 Pipette Man
B-3.2 Reagents
B-3.2.1 Cetyl Trimethyl Ammonium Bromide (CTAB)
Buffer
CTAB : 1 percent
Sodium Dodecyl Sulphate (SDS) : 1 percent
EDTA : 20 mM
Sodium Chloride : 1 M
B-3.2.2 Phenol, Chloroform and Isoamylalcohol in the
ratio of 25:24:1 (PCI)
B-3.2.3 Ethanol
B-3.2.4 TE Buffer (pH 8.0)
Tris base : 1 M
EDTA : 0.5 M
B-3.2.5 Sodium Acetate — 3 M.
11. 8
IS 10500 : 2012
B-3.3 Procedure
Treat 300 µl of concentrated water sample with equal
volume of CTAB and 1/10th volume of PCI. Vortex
and centrifuge at 5 000 × g for 30 min at 4°C. Add 1/
10th volume of 3 M sodium acetate and double the
volume of cold ethanol to the aqueous layer. Keep the
mixture at either at –20°C for overnight or in liquid
nitrogen for 2-5 min. Centrifuge at 10 000 × g, for
30 min at 4°C. Discard the supernatant and air dry the
pellet and dissolve it in 20 µl TE buffer.
B-4 COMPLEMENTARYDNA(c DNA)SYNTHESIS
B-4.1 Apparatus
B-4.1.1 PCR Machine
B-4.1.2 Deep Freezer (–20°C)
B-4.2 Reagents
B-4.2.1 cDNA Synthesis Kit
B-4.3 Procedure
Suspend the extracted RNA in 20 µl of cDNA reaction
mixture, which consists of 4 µl of 5X reverse
transcriptase reaction buffer [250 mM TRIS–HCl
(pH 8.5), 40 mM KCl, 150 mM MgCl2, 5 mM
dithiothreitol (DTT)], 0.5 µl of 10 mM deoxynucleotide
phosphate (dNTP), 2 µl of hexa nucleotide mixture,
1 µl of 25 U of Maloney Murine Leukaemia Virus (M-
MuLV) reverse transcriptase, 0.5 µl of 20 U of human
placental RNase inhibitor. Heat the reaction mixture to
95°C for 5 min and rapidly chill on ice, this is followed
by the addition of 1 µl (25 U/µl) of M-MuLV reverse
transcriptase. Incubate the reaction mixture as given
by the manufacturer of the kit and quickly chill the
reaction tube on ice.
B-5 PCR AMPLIFICATION
B-5.1 Apparatus
B-5.1.1 PCR Machine
B-5.1.2 Deep Freezer (–20°C)
B-5.1.3 Micropippette
B-5.2 Reagents
B-5.2.1 Primers for EV and HAV
EV sense primer, 5’ — TCC TCC GGC CCC
TGA ATG CG — 3’
antisense primer, 5’ — ATT GTC ACC
ATA AGC AGC CA — 3’
HAV sense primer, 5’ — GTTTT GCTCC
TCTTT ATCAT GCTAT G-3’
antisense primer, 5’ — GGAAA TGTCT
CAGGT ACTTT CTTTG-3’
B-5.2.2 PCR Master Mix
B-5.2.3 Mineral Oil
B-5.3 Procedure
B-5.3.1 PCR Amplification for Hepatitis AVirus (HAV)
In 5 µl of cDNA, add 95 µl of a PCR Master Mix (10
mM TRIS–HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2,
0.01 percent gelatin (1× PCR buffer), 200 µM of each
dNTP, 1.5 U of Thermus aquaticus polymerase). Add
25 pico moles of sense and antisense oligonucleotide
primers of HAV and overlay with mineral oil.
Appropriate positive and negative controls shall be
included with each run. Set the following reaction at
thermo cycler:
Denaturation at 94°C for 2 min
Denaturation for 1.0 min at 94°C
Annealing for 1.0 min at 57°C
Extension for 1.3 min at 72°C
Final extension at 72°C for 7 min.
B-5.3.2 PCR Amplification for Enterovirus (EV)
In 5 µl of cDNA, add 95 µl of a PCR Master Mix (10
mM TRIS–HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2,
0.01 percent gelatin (1X PCR buffer), 200 µM of each
dNTP, 1.5 U of Thermus aquaticus polymerase). Add
25 pico moles of sense and antisense oligonucleotide
primers of EV and overlay with mineral oil.
Appropriate positive and negative controls shall be
included with each run. Set the following reaction at
thermo cycler:
Denaturation at 94°C for 2 min
Denaturation for 1.0 min at 94°C
Annealing for 1.0 min at 42°C
Extension for 2.0 min at 72°C
Final extension at 72°C for 7 min.
B-6 AGAROSE GEL ELECTROPHORESIS
B-6.1 Apparatus
B-6.1.1 Micropippette
B-6.1.2 Electrophoresis Apparatus
B-6.1.3 Gel Documentation System
B-6.2 Reagents
B-6.2.1 Running Buffer — 50X TAE buffer
Tris base/Tris buffer : 121.00 g
35 cycles
35 cycles
12. 9
IS 10500 : 2012
Glacial acetic acid : 28.55 ml
0.5 M EDTA : 50 .00 ml
Distilled water : 300.45 ml
(autoclaved)
Make the final volume upto 1 000 ml with deionised
distilled water, sterilize and store at 4°C. The final
concentration for the preparation of agarose gel and to
run the gel shall be 1X.
B-6.2.2 Tracking Dye — 6X bromophenol blue.
B-6.2.3 Ethidium Bromide — 0.5 µg/ml.
B-6.3 Procedure
Run the PCR amplified product of EV and HAV on
1.5 percent agarose gel using 1X TAE buffer. Load
10 µl of amplified product after mixing it with 1 µl
10X loading dye. Run the molecular weight marker
along with the samples. Run the electrophoresis at
100 V for 30 min. Stain the gel with ethidium bromide
(0.5 µl/ml) for 20 min. Wash it with distilled water
and view under UV transilluminator and photograph
the gel to analyse the band pattern. EV gives the band
as 155 base pair and the HAV gives band as 225 base
pair.
ANNEX C
(Clause 4.3.10)
ILLUSTRATIVE LIST OF MICROSCOPIC ORGANISMS PRESENT IN WATER
Sl
No.
Classification of
Microscopic
Organism
Group and Name of the Organism Habitat Effect of the
Organisms and
Significance
(1) (2) (3) (4) (5)
i) Algae a) Chlorophyceae:
1) Species of Coelastrum, Gomphospherium,
Micractinium, Mougeotia, Oocystis,
Euastrum, Scenedesmus, Actinastrum,
Gonium, Eudorina Pandorina, Pediastrum,
Zygnema, Chlamydomonas, Careteria,
Chlorella, Chroococcus, Spirogyra,
Tetraedron, Chlorogonium, Stigeoclonium
Polluted water,
impounded
sources
Impart
colouration
2) Species of Pandorina, Volvox,
Gomphospherium, Staurastrum,
Hydrodictyon, Nitella
Polluted waters Produce taste and
odour
3) Species of Rhizoclonium, Cladothrix,
Ankistrodesmus, Ulothrix, Micrasterias,
Chromulina
Clean water Indicate clean
condition
4) Species of Chlorella, Tribonema,
Clostrium, Spirogyra, Palmella
Polluted waters,
impounded
sources
Clog filters and
create impounded
difficulties
b) Cyanophyceae:
1) Species of Anacystis and Cylindrospermum Polluted waters Cause water
bloom and impart
colour
2) Species of Anabena, Phormidium,
Lyngbya, Arthrospira, Oscillatona
Polluted waters Impart colour
3) Species of Anabena, Anacystis,
Aphanizomenon
Polluted waters,
impounded
sources
Produce taste and
odour
4) Species of Anacystis, Anabena,
Coelospherium, Cleotrichina,
Aphanizomenon
Polluted waters Toxin producing
5) Species of Anacystis, Rivularia,
Oscillatoria, Anabena
Polluted waters Clog filters
13. 10
IS 10500 : 2012
Sl
No.
Classification of
Microscopic
Organism
Group and Name of the Organism Habitat Effect of the
Organisms and
Significance
(1) (2) (3) (4) (5)
6) Species of Rivularia Calcareous
waters and also
rocks
Bores rocks and
calcareous strata
and causes
matted growth
7) Species of Agmenellum, Microcoleus,
Lemanea
Clean waters Indicators of
purification
c) Diatoms (Bacillareophyceae):
1) Species of Fragillaria, Stephanodiscus,
Stauroneis
— Cause
discoloration
2) Species of Asterionella, Tabellaria Hill streams
high altitude,
torrential and
temperate waters
Taste and odour
producing clog
filters
3) Species of Synedra and Fragillavia Polluted waters Taste and odour
producing
4) Species of Nitzchia, Gomphonema Moderately
polluted waters
Cause
discoloration
5) Species of Cymbela, Synedra, Melosira,
Navicula, Cyclotella, Fragillaria, Diatoma,
Pleurogsigma
Rivers and
streams
impounded
sources
Clog filters and
cause operational
difficulties
6) Species of Pinmularia, Surinella,
Cyclotella, Meridion, Cocconeis
Clean waters Indicators of
purification
d) Xanthophyceae:
Species of Botryococcus Hill streams,
high altitude and
temperate waters
Produces
coloration
ii) Zooplankton a) Protozoa:
1) Amoeba, Giardia Lamblia Arcella,
Difflugia, Actinophrys
Polluted waters Pollution
indicators
2) Endamoeba, Histolytica Sewage and
activated sludge
Parasitic and
pathogenic
b) Ciliates:
Paramoecium, Vorticella, Carchesium,
Stentor, Colpidium, Coleps, Euplotes,
Colopoda, Bodo
Highly polluted
waters, sewage
and activated
sludge
Bacteria eaters
c) Crustacea:
1) Bosmina, Daphnia Stagnant pollu-
ted waters
Indicators of
pollution
2) Cyclops Step wells in
tropical climate
Carrier host of
guinea worm
iii) Rotifers a) Rotifers:
Anurea, Rotaria, Philodina Polluted and
Algae laden
waters
Feed on algae
b) Flagellates:
1) Ceratium, Glenodinium, Peridinium
Dinobryon
Rocky strata, iron
bearing and
acidic waters
Impart colour
and fishy taste
2) Euglena, Phacus Polluted waters Impart colour
14. 11
IS 10500 : 2012
Sl
No.
Classification of
Microscopic
Organism
Group and Name of the Organism Habitat Effect of the Organisms
and Significance
(1) (2) (3) (4) (5)
iv) Miscellaneous
Organisms
a) Sponges, Hydra Fresh water Clog filters and affect
purification systems
b) Tubifex, Eristalls, Chironomids Highly polluted waters,
sewage and activated
sludge and bottom
deposits
Clog filters and render
water unaesthetic
c) Plumatella Polluted waters Produces biological
slimes and causes filter
operational difficulties
c) Dreissena, Asellus Polluted waters Harbour pathogenic
organisms
15.
16. Bureau of Indian Standards
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Review of Indian Standards
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should ascertain that they are in possession of the latest amendments or edition by referring to the latest issue of
‘BIS Catalogue’ and ‘Standards : Monthly Additions’.
This Indian Standard has been developed from Doc No.: FAD 25 (2047).
Amendments Issued Since Publication
Amend No. Date of Issue Text Affected
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Published by BIS, New Delhi
